This Phase I research proposal is to investigate the feasibility of developing a novel Biomarker Breast PapTest for screening Nipple Aspirate Fluid (NAF) and detecting breast cancers at the tumor-in-formation stage before mammographically detectable tumors are developed. Although there is a slight decline in deaths from breast cancer in recent years, it continues to be the most diagnosed cancer and the second leading cause of cancer deaths for women in the USA. We can expect a drastic reduction in the incidence and deaths if high risk women are identified before tumor formation and treated prophylactically. However, precise identification of high risk women for prophylactic measures has been a major clinical challenge. Currently, risk is assessed by Gail scoring and cytological examination of ductal cells in NAF elicited using NeOMatrix instrument (HALO Breast Pap Test). However, numerous studies have shown that cytology based risk assessment is highly subjective and not precise enough to undertake prophylactic treatments. Several arbitrarily selected markers were explored in NAF cells as risk predictors, but none established thus far. Our long range goal is to reduce breast cancer incidence by identifying high risk women in the absence of detectable tumors. In Phase I we plan to investigate several markers for test development that were previously shown to accurately predict risk in patients with premalignant tumors. Our aims include identifying potential markers by screening NAF from subjects with cancer, ADH, Benign tumors and healthy subjects. In Phase II our aims include screening large number of NAF samples and correlating the expression of multiple markers with tumor development, multimarker Test algorithm building and validation. The proposed work is innovative, because risk assessment will be based on the expression of multiple cancer promoting molecules in NAF and capitalizes on our previously established risk prediction markers in premalignant patients. It is our expectation that expression of cancer markers in NAF will precisely predict breast cancer risk in any woman irrespective of cytology, family history or any other conventional risk factors in the absence of detectable tumors. The outcomes will be significant because once the test is developed and validated it could be applied to screen any women in the absence of mammographically or MRI detectable tumors and precisely predict breast cancer risk. The outcomes will be a major break-though in molecular risk prediction for breast cancer. Treatment before tumor formation and prevention will become a reality. It is our expectation that the outcomes will have a major impact in drastically reducing the incidence and deaths from breast cancer, simultaneously saving Billions of dollars in Health Care Cost.